WO2017213588A1 - Procédé et trousse de détection et/ou de quantification d'endotoxine - Google Patents

Procédé et trousse de détection et/ou de quantification d'endotoxine Download PDF

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WO2017213588A1
WO2017213588A1 PCT/SG2017/050287 SG2017050287W WO2017213588A1 WO 2017213588 A1 WO2017213588 A1 WO 2017213588A1 SG 2017050287 W SG2017050287 W SG 2017050287W WO 2017213588 A1 WO2017213588 A1 WO 2017213588A1
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endotoxin
lps
labeled
labeling agent
mixture
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PCT/SG2017/050287
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English (en)
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Huatao FENG
Min SU
Fun Man FUNG
Fong Yau Sam LI
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National University Of Singapore
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
    • G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • G01N2400/50—Lipopolysaccharides; LPS

Definitions

  • Various embodiments relate to a method for detecting and/or quantifying endotoxin, and to a kit for detecting and/or quantifying endotoxin.
  • Endotoxins also called lipopolysaccharides (LPS)
  • LPS lipopolysaccharides
  • endotoxins are major constituents of an outer membrane of Gram- negative bacteria.
  • endotoxin is usually used to refer to LPSs that are attached on the bacterial membrane, while the term “LPS” is usually used to refer to the purified form.
  • Dry weight of LPS constitutes about 5 to 10 % of a bacterial cell, with LPS molecules occupying approximately three quarters of the surface area of the outer membrane of Escherichia coli, with the remaining area occupied by certain proteins.
  • the outer membrane of a bacterial cell serves as a barrier for environmental stresses and various toxic substances such as antibiotics, it also functions as a nutrient transporter. Accordingly, LPSs released from the bacterial surface during growth, cell division and lysis, may translate into universal toxic inflammatory agents in environment.
  • Endotoxins are toxic because they can influence many cellular and humoral mediated systems.
  • LPSs in the blood stream of an infected host may induce fever, diarrhea, vomiting, cough, breathing difficulties, shock, leukocytosis, intravascular coagulation, multi organ failure, septic shock, and even death.
  • Even 1 to 10 ng (10 to 100 EU)/kg body weight (intravenously) of endotoxin can induce fever.
  • endotoxins can augment the toxicity of some chemicals such as microcystins. Endotoxins have been considered as the main cause of pyrogenic reactions that may take place during the administration of bio therapeutics.
  • LPS are complicated macromolecules which exhibit heterogeneous structure within and between strains.
  • LPS complex amphiphilic molecular structures
  • LPS may vary among different bacterial strains, they share a common architecture typically consisting of a polysaccharide part and a lipid component.
  • the polysaccharide part is covalently bound to the lipid component termed "lipid A".
  • Structure of the hydrophobic glucosamine-based lipid A is conserved during the biochemical synthesis.
  • the lipid A is responsible for biological activity and toxicity of the LPS, while the polysaccharide part is responsible for antigenicity of the LPS .
  • the polysaccharide part may be sub-divided into two regions of a core oligosaccharide region and O-antigen polysaccharide.
  • LPS may comprise the following three main regions: lipid A, core oligosaccharide, and O-antigen polysaccharide.
  • the core oligosaccharide region which is usually identical for large groups of bacteria, may consist of two or three 2-keto-3-deoxy-octonic acids (KDOs) and 8 to 15 monosaccharides.
  • KDOs 2-keto-3-deoxy-octonic acids
  • the O-antigen polysaccharide otherwise termed as O-antigen, responsible for antigenicity and serotype-specific immunogenicity of the LPS, may be composed of various numbers of repeating oligosaccharide subunits due to different biosynthesis.
  • LPS monomers have a molecular weight of approximately 10 to 20 kDa, although in some cases, molecular weight of LPS monomers may range from 2 to 30 kDa. Due to its amphipathic structures, however, LPS molecules have strong tendency to form aggregates into vesicles in aqueous solution with a micellar weight around one million daltons.
  • the smooth-type LPSs which contain all the three components in the structure, can usually be isolated from the wild-type bacterial strains. Absence of the O-specific region (due to mutation) results in a shortened and more hydrophobic bacterial strain, with a "rough" colony morphology, normally called rough-type LPSs.
  • LAL assay is extremely sensitive with detection limits as low as 100 picograms per milliliter, it has many shortcomings. For example, effects of temperature, pH, ions, and even reagent chemicals can influence the LAL results. The non-specific methods would also give false positive results due to reactions with other microbial products that trigger the LAL reaction, such as peptidoglycan from gram-positive organisms, (1-3)- ⁇ - ⁇ - glucans, and D-glucose polymers released from cell walls. Furthermore, the LAL method only provides the general activity of endotoxins in the sample, instead of the detailed structure or distribution profile.
  • LAL method measures endotoxin concentration indirectly, while endotoxins possess diverse structures and activities that originate from different bacterial serotypes and their mutants may introduce variations. These structural variations have a direct impact on endotoxin toxicity and endotoxin reactivity in the LAL test. Moreover, endotoxins are able to form agglutinate or micelles. These micellar structures reduce their reactivity relative to LAL and make LAL result inaccurate to reflect the true concentration of endotoxins. A reliable analytical method for endotoxin analysis will serve as a useful tool in various industries such as drinking water purification processes and water reclamation plants.
  • a method for detecting and/or quantifying endotoxin comprises
  • kits for detecting and/or quantifying endotoxin comprises
  • a labeling agent capable of binding to endotoxin comprised in a sample suspected of containing endotoxin to form a labeled endotoxin
  • an enriching medium capable of enriching the labeled endotoxin in the sample.
  • FIG. 1 is a graph showing fluorescein isothiocyanate labeled LPS (FITC-LPS) (from E. Coli 0111:B4J separated in Na 2 B 4 07 buffer of different concentrations (1) 10 mM; (2) 20 mM; (3) 50 mM; (4) 60 mM; (5) 80 mM. Other conditions: Bare fused silica capillary, (I.D. 50 ⁇ ). Total length: 39 cm; Voltage: 30 kV; Sample storage: 25 °C; Sample injection time: 5 s.
  • FITC-LPS fluorescein isothiocyanate labeled LPS
  • FIG. 2 is a graph showing FITC-LPS (from Serratia marcescens) separated in sodium tetraborate buffer (50 mM) with different pH values: (1) 8.50; (2) 9.00; (3) 9.25; (4) 9.30; (5) 9.70; (6) 10.0.
  • Other conditions Bare fused silica capillary (I.D. 50 ⁇ ); Total length: 39 cm; Voltage: 30 kV; Sample storage: 25 °C; Sample injection time: 5 s.
  • FIG. 3 is a graph showing LPS from Salmonella typhosa separated in 50 mM Na 2 B 4 0 7 buffer using different capillary lengths of 38.8 cm, 48.8 cm, and 80.0 cm. Other conditions: Separation buffer: Na 2 B 4 0 7 (50 mM), pH 9.30; Bare fused silica capillary, (I.D. 50 ⁇ ); Total length: 39 cm; Voltage: 30 kV; Sample storage: 25 °C; Sample injection time: 5 s.
  • FIG. 4 is a graph showing capillary electrophoresis and laser-induced fluorescence (CE-LIF) separation of 13 endotoxins and free FITC as reference.
  • LPS concentration 500 ⁇ g/mL. Separation in buffer: Na 2 B 4 0 7 (50 mM), pH 9.30; Bare fused silica capillary, (I.D. 50 ⁇ ); Total length: 39 cm; Voltage: 30 kV; Sample storage: 25 °C; Sample injection time: 5 s.
  • FIG. 5 is a graph depicting stability study of LPS-FITC from Day 1 to Day 20. Other conditions: Separating in buffer: Na 2 B 4 0 7 (50 mM), pH 9.30; Bare fused silica capillary, (I.D. 50 ⁇ ); Total length: 39 cm; Voltage: 30 kV; Sample storage: 25 °C; Sample injection time: 5 s.
  • FIG. 6 is a graph depicting analysis of FITC labeled LPS extracted by WATERS HILIC ⁇ plate. Separating in buffer: Na 2 B 4 07 (50 mM), pH 9.30; Bare fused silica capillary, (I.D. 50 ⁇ ). Total length: 39 cm; Voltage: 30 kV; Sample amount: 100 ⁇ ; Sample storage: 25 °C; Sample injection: 5 s.
  • FIG. 7 is a graph showing HILIC ⁇ plate to remove excess FITC dye from labeled LPS from Klebsiella pneumoniae. Separating in buffer: Na 2 B 4 07 (50 mM), pH 9.30; bare fused silica capillary, (I.D. 50 ⁇ ). Total length: 39 cm; Voltage: 30 kV; Sample storage: 25 °C; Sample injection: 5 s.
  • FIG. 8 is a graph showing (l-3)-P-D-glucans interference test.
  • FIG. 9 shows loss of short R-type endotoxin with solid phase extraction (SPE) extraction procedures (If oligosaccharide was shorter than Rc mutant).
  • CE conditions Separation buffer: Na 2 B 4 0 7 (50 mM), pH 9.30; Bare fused silica capillary, (I.D. 50 ⁇ ); Total length: 39 cm; Voltage: 30 kV; Sample storage: 25 °C; Sample injection time: 5 s.
  • FIG. 10 is a graph depicting investigation of detection limit of endotoxin from Klebsiella Pneumonia by ultrapure water dilution. Other conditions: Separating in buffer: Na 2 B 4 O 7 (50 mM), pH 9.30; Bare fused silica capillary, (I.D. 100 ⁇ ); Total length: 60 cm; Voltage: 15 kV; Sample injection time: 10 s.
  • FIG. 11 is a graph showing analysis of LPS level in raw effluent and tap water.
  • CE conditions Separating in buffer: Na 2 B 4 0 7 (50 mM), pH 9.30; Bare fused silica capillary, (I.D. 50 ⁇ ); Total length: 39 cm; Voltage: 30 kV; Sample storage: 25 °C; Sample injection time: 5 s.
  • FIG. 12 is a graph depicting detection of FITC labeled LPS from Serratia Marcescens using borate buffer.
  • FIG. 13 is a graph depicting separation of Alexa 488 hydrazide-LPS (from Salmonella enterica serotype minnesota) separating in borate buffer.
  • FIG. 14 is a graph showing good linearity of CE-LIF method using FITC labeling.
  • FIG. 15 is a graph showing LPS from Salmonella enterica serotype Minnesota. Red curve: LPS before SPE. Blue curve: LPS after PGC SPE.
  • FIG. 16 is a graph showing LPS from Salmonella enterica serotype Minnesota. Red curve: LPS before SPE. Blue curve: LPS after Hilic SPE.
  • FIG. 17 is a graph showing that good linearity of LPS from E. Coli 026:B6 was observed with Hilic SPE.
  • FIG. 18 shows endotoxin collected after lipid extraction device was labeled by Alexa Fluor 488. Red curve: 0128:B 12-control; Blue curve: 0128:B 12-SPE.
  • FIG. 19A through FIG. 19C depicts a method according to an embodiment disclosed herein.
  • a sample suspected of containing endotoxin 101 is contacted with a labeling agent 102 to form a mixture, wherein the contacting is carried out under conditions sufficient to bind the labeling agent 102 to the endotoxin 101 to form a labeled endotoxin 103 (FIG. 19B).
  • the mixture is enriched for the labeled endotoxin 103 by an enrichment process 100 such as solid phase extraction to remove impurities 104.
  • a signal is detected from the enriched mixture using an analytical method 110 such as capillary electrophoresis and laser induced fluorescence (CE-LIF) detection for indication of the presence of endotoxin as shown in FIG. 19C.
  • CE-LIF laser induced fluorescence
  • Various embodiments refer in a first aspect to a method for detecting and/or quantifying endotoxin.
  • the various embodiments described in the context of the method are analogously valid for or applicable to a kit for detecting and/or quantifying endotoxin.
  • the present method and kit are able to detect and/or quantify endotoxins.
  • accurate, reliable, and comparable information on level and type of endotoxins in samples may be obtained based on analytical methodologies, such as capillary electrophoresis (CE) and laser induced fluorescence (LIF) detection.
  • capillary electrophoresis (CE) with laser induced fluorescent (LIF) detection for the analysis of intact endotoxin molecules has not previously been studied.
  • the sample may be subjected to a pre- treatment step, such as solid phase extraction (SPE), to extract endotoxins from interference materials.
  • a pre- treatment step such as solid phase extraction (SPE)
  • SPE solid phase extraction
  • endotoxins By enriching endotoxins using the pre-treatment step, sensitivity of the analysis may be increased.
  • the method may comprise contacting a sample suspected of containing endotoxin with a labeling agent to form a mixture, wherein the contacting is carried out under conditions sufficient to bind the labeling agent to the endotoxin to form a labeled endotoxin, enriching the mixture for the labeled endotoxin, and detecting a signal from the enriched mixture as indication of the presence of endotoxin.
  • sample suspected of containing endotoxin may refer to any liquid, such as body fluid, water, or waste effluent, which is suspected of containing endotoxin.
  • the endotoxin that is suspected to be present in the sample may be of any suitable amount or concentration, as the method disclosed herein is effective in detecting endotoxins at very low detection limits of sub-ppb levels, such as around 1 ng/ mL.
  • Contacting the sample suspected of containing endotoxin with the labeling agent may be carried out by, for example, dispersing the labeling agent into the sample. Dispersing the labeling agent into the sample may be carried out under physical agitation such as stirring or sonication. The contacting may be carried out under conditions sufficient to bind the labeling agent to the endotoxin to form a labeled endotoxin, which may take place at various temperatures and pH levels, and which may be carried out for various time periods.
  • contacting the sample suspected of containing endotoxin with the labeling agent may be carried out at a temperature in the range of about 30 °C to about 45 °C, such as about 35 °C to about 45 °C, about 40 °C to about 45 °C, about 30 °C to about 40 °C, about 30 °C to about 35 °C, or about 35 °C to about 40 °C.
  • contacting the sample suspected of containing endotoxin with the labeling agent is carried out at a temperature of about 37 °C.
  • contacting the sample suspected of containing endotoxin with the labeling agent may be carried out at ambient temperature, defined herein as a temperature in the range of about 30 °C to about 35 °C.
  • ambient temperature defined herein as a temperature in the range of about 30 °C to about 35 °C.
  • heating of the sample may not be required in these embodiments.
  • contacting the sample suspected of containing endotoxin with the labeling agent may be carried out at alkaline pH, such as a pH of greater than 8.5, greater than 9, greater than 9.5, or greater than 10.
  • alkaline pH such as a pH of greater than 8.5, greater than 9, greater than 9.5, or greater than 10.
  • contacting the sample suspected of containing endotoxin with the labeling agent is carried out at a pH in the range of about 8.5 to about 11.5, such as about 9.5 to about 11.5, about 10 to about 11.5, or about 10.5 to about 11.5.
  • contacting the sample suspected of containing endotoxin with the labeling agent is carried out at a pH in the range of about 10 to about 11.
  • contacting the sample suspected of containing endotoxin with the labeling agent may be carried out for a time period in the range of about 1 hour to about 6 hours, such as about 2 hours to about 6 hours, about 3 hours to about 6 hours, or about 1 hour to about 5 hours.
  • contacting the sample suspected of containing endotoxin with the labeling agent is carried out for a time period in the range of about 2 hours to about 4 hours.
  • contacting the sample suspected of containing endotoxin with the labeling agent within the temperature range, pH range, and time period specified above may provide optimized separation conditions, which translate into rapid analysis and accurate detecting of LPS during subsequent analysis.
  • contacting the sample suspected of containing endotoxin with the labeling agent is carried out for a time period in the range of about 2 hours to about 4 hours at a temperature of about 37 °C.
  • the sample suspected of containing endotoxin is contacted with a labeling agent.
  • labeling agent refers to a molecule, compound or reagent that may be operatively linked or bound to endotoxin, so as to allow detection of the endotoxin when the labeling agent is detected.
  • the binding of the labeling agent to the endotoxin may take place via a functional group such as amino group that is present on the endotoxin.
  • the labeling agent covalently binds to the endotoxin at the lipid A portion and/or core portion of the endotoxin.
  • the endotoxin By binding the labeling agent to the endotoxin, the endotoxin may be "tagged" by the labeling agent to allow its detection by subsequent analysis.
  • the lipid A and the core domains show greatest similarity among various bacterial strains. As such, labeling this part of the endotoxin would constitute a most general strategy for endotoxin detection.
  • the labeling agent may accordingly be any suitable moiety that may be operatively linked or bound to endotoxin, and which may be readily detected using an assay method.
  • the labeling agent comprises a fluorescent dye.
  • fluorescent dye refers to a substance or compound which is capable of emitting light when excited by another light of appropriate wavelength.
  • the fluorescent dye is capable of emitting light when excited by light having a wavelength in the range of about 400 nm to about 500 nm.
  • the fluorescent dye may be selected from the group consisting of fluorescein isothiocyanate (FITC), fluorescamine, FMOC, FQ, CBQCA, NanoOrange, Alexa 488 Hydrazide, NDA, Nile red, 5-DTAF, syto 9, syto 13, YO- PRO-1, TAMRA, RITC, Funl cell stain, and combinations thereof.
  • FITC fluorescein isothiocyanate
  • fluorescamine fluorescamine
  • FMOC FMOC
  • FQ fluorescamine
  • CBQCA NanoOrange
  • Alexa 488 Hydrazide NDA
  • Nile red Nile red
  • 5-DTAF syto 9
  • syto 13 YO- PRO-1
  • TAMRA RITC
  • Funl cell stain and combinations thereof.
  • the labeling agent comprises or consist of fluorescein isothiocyanate isomer 1 (FITC). It has high quantum yield (0.92) and may be used as a fluorescent derivatizing agent for analysis of amino-group containing analytes such as endotoxin, in embodiments where detection is carried out using laser induced fluorescent detection.
  • FITC fluorescein isothiocyanate isomer 1
  • the labeling agent used may at least be of the same amount to, or be in excess to the amount of endotoxin that is suspected to be present in the sample. This is to ensure that all of the endotoxins suspected to be present in the sample are tagged with the labeling agent.
  • weight ratio of the labeling agent to the endotoxin suspected to be present in the sample may be in the range of about 1: 1 to about 15: 1, such as about 2: 1 to about 15: 1, about 3: 1 to about 15: 1, about 5: 1 to about 15: 1, or about 2: 1 to about 10: 1. In some embodiments, weight ratio of the labeling agent to the endotoxin is about 3: 1.
  • the method disclosed herein may comprise enriching the mixture for the labeled endotoxin.
  • enriching refers to selective accumulation or increasing concentration of the labeled endotoxin in the mixture. Enriching the mixture for the labeled endotoxin may be carried out using any suitable method, for example, by removing impurities such as free labeling agent from the mixture.
  • enriching the mixture for the labeled endotoxin in various embodiments comprises removing free labeling agent from the mixture.
  • the term "free labeling agent” refers to any labeling agent that is not bound to an endotoxin. This removal may be achieved, for example, by any means suitable for separating the labeled endotoxins from the free labeling agents.
  • enriching the mixture for the labeled endotoxin is carried out using a method selected from the group consisting of solid phase extraction, carbohydrate extraction, lipid extraction, and combinations thereof.
  • enriching of the mixture for the labeled endotoxin is carried out using solid phase extraction.
  • solid-phase extraction refers to a separation technique which is based on the affinity of solutes dissolved or suspended in a liquid (known as the mobile phase) for a solid through which the liquid is passed (known as the stationary phase or sorbent), so as to separate a mixture into components of interest and other components. The result is that the desired analytes are separated from the mixture and the analytes may reside either in the mobile phase or in the stationary phase.
  • enriching of the mixture for the labeled endotoxin may comprise absorbing the labeled endotoxin on a sorbent in the mixture, and extracting the labeled endotoxin from the sorbent using a solvent. In so doing, at least some of, or most of the interference substances may be removed so as to reduce or remove undesirable background signals.
  • any liquid which may be used to extract or elute the labeled endotoxin from the sorbent may be used. Examples may include, but are not limited to, an aqueous liquid such as water, and/or a buffer solution such as tris-citrate buffer, phosphate buffer, boric buffer, and/or zwitterionic buffer.
  • peptide or protein may be labeled by FITC, they may be removed using the above enriching procedure by not being absorbed on the sorbent.
  • polysaccharides may be absorbed by the sorbent, they will not be detected by subsequent analysis such as using the CE-LIF method since they are not labeled by FITC.
  • the sorbent may accordingly be any suitable material that is able to selectively absorb labeled endotoxin and may, for example, be selected from the group consisting of a solid phase extraction (SPE) packing material, silica-based aminopropyl sorbent, porous graphite column (PGC), carboxyl-coated magnetic beads, and combinations thereof.
  • SPE solid phase extraction
  • PLC porous graphite column
  • the sorbent comprises or consists of silica-based aminopropyl sorbent.
  • Silica-based aminopropyl sorbent has the capacity to strongly and selectively retain analytes that are hydrophilic in nature.
  • carbohydrate is one of the main components in the endotoxin structure, and which is hydrophilic, retention of endotoxin onto the silica-based aminopropyl sorbent may take place due to hydrogen bonding, as well as ionic and dipole-dipole interactions that occur while the carbohydrates partition into an immobilized water layer.
  • the method disclosed herein may comprise detecting a signal from the enriched mixture as indication of the presence of endotoxin.
  • Detection of a signal from the enriched mixture may be carried out using any suitable analytical method involving procedures, such as sample preparation, separation, and detection, which are similar to the procedures outlined herein.
  • suitable analytical methods include, but are not limited to, capillary electrophoresis and laser-induced fluorescence (CE-LIF) detection, high-performance liquid chromatography (HPLC) with fluorescent detection, ion chromatography (IC) with fluorescent detection, and capillary electrophoresis with normal light source detection.
  • capillary electrophoresis is a highly sensitive, high resolution analytical technique for the separation, characterization, and quantification of analytes, which may be used in conjunction with laser induced fluorescence (LIF) technique to analyze intact endotoxin molecules.
  • LIF laser induced fluorescence
  • Capillary electrophoresis may generally involve application of high voltages across buffer-filled capillaries to achieve separations.
  • the capillaries used are usually fused silica capillaries covered with an external polymer protective coating to give them increased mechanical strength.
  • Capillaries may be about 25 cm to 100 cm long, and have an inner diameter of about 50 to 100 ⁇ .
  • the capillary may be filled with a buffer solution which conducts current through the inside of the capillary. The ends of the capillary are dipped into reservoirs filled with the buffer. Electrodes made of an inert material such as platinum may also inserted into the buffer reservoirs to complete the electrical circuit.
  • the buffer solution should not comprise a reagent and/or additive containing reactive functional groups such as amino groups, which may otherwise bind to the labeling agent.
  • tris(hydroxymethyl)aminomethane (tris) and 1,4-diaminobutane (DAB), for example are not suitable candidates for use as the buffer solution disclosed herein.
  • the buffer solution may, for example, be selected from the group consisting of borate, phosphate, bicarbonate, triethanolamine (TEA), bicine (N,N-Bis-(2- hydroxyethyl)-glycine), (2-(N-Cyclohexylamine)-ethanesulfonic acid) (CHES), (3- Cyclohexylamino)-l-propanesulfonic acid) (CAPS), glycine, (N-[Tris-(hydroxymethyl)- methyl] -glycine) (Tricine), and combinations thereof.
  • TAA triethanolamine
  • bicine N,N-Bis-(2- hydroxyethyl)-glycine
  • CHES (2-(N-Cyclohexylamine)-ethanesulfonic acid)
  • CAS (3- Cyclohexylamino)-l-propanesulfonic acid)
  • Tricine Tricine
  • the buffer solution comprises or consists of borate.
  • endotoxins being neutral compounds, which makes them unsuitable for electrophoretic analysis
  • the inventors have surprisingly found that complexation of borate ions (B(OH) 4 ⁇ ) with cis-diol groups in the carbohydrate moiety within the endotoxin structure enhances electrophoretic mobilities of endotoxin, as borate complexation provides the uncharged glycolipid with negative charges thus making them mobile in capillary electrophoresis analysis.
  • Separation efficiency using capillary electrophoresis may be affected by concentration of the buffer solution.
  • concentration of the buffer solution Generally, increasing concentration of the buffer solution increases ionic strength which translates into increase in migration time due to decreased electroosmotic flow (EOF).
  • EEF electroosmotic flow
  • peaks that is generated from analysis may not be well separated.
  • concentration of the buffer solution may generally be in the range of about 10 mM to about 100 mM, such as about 30 mM to about 100 mM, about 40 mM to about 60 mM, about 10 mM to about 80 mM, or about 50 mM.
  • concentration of about 40 mM to about 60 mM or about 50 mM provides an optimum buffer concentration with the highest fluorescence intensity and satisfactory resolution.
  • pH of the buffer solution may be in the range of about 8 to about 1 1, such as about 8.5 to about 10, about 9 to about 9.8, about 9 to about 9.5, or about 9.3.
  • at least a portion of the endotoxins may acquire a negative charge, thereby facilitating complexation with ions, such as borate ions (B(OH) 4 ⁇ ), that may be present in the buffer solution.
  • ions such as borate ions (B(OH) 4 ⁇ )
  • capillary electrophoresis is able to provide good separation and laser-induced fluorescence detection allows detection of minute amounts of the labeled endotoxin.
  • methods disclosed herein may avoid possible false positive results commonly associated with state of the art LAL assays, due to the reaction with (l-3)-P-D-glucans as these interference substances will trigger the LAL reaction.
  • the methods disclosed herein are able to provide inherent discrimination of interfering substances including (l-3)-P-D-glucans due to the specificity of the derivatizing agent.
  • the laser induced fluorescence may be carried out using an excitation source such as a UV lamp, LED lamp, and/or laser. Wavelength of the excitation source may be in the range of about 400 nm to about 500 nm, and emission wavelength may range from about 490 nm to about 560 nm. In various embodiments, the laser induced fluorescence (LIF) is carried out at excitation wavelength of 488 nm and emission wavelength of 520 nm to generate fluorescence spectra.
  • Use of laser induced fluorescence compares favorably against other analysis methods such as UV absorbance detection since endotoxin lacks natural optically active groups in its chemical structure, which makes its detection by UV absorbance detector a challenge.
  • a labeling agent such as a fluorescent dye to associate or label the endotoxin
  • use of laser induced fluorescence may be enabled as endotoxins may not possess native fluorescence properties.
  • detecting a signal from the enriched mixture may comprise generating a signal spectrum and detecting at least one of changes in pattern and/or intensity in the signal spectrum; and shifts in the signal spectrum as an indication of the presence of endotoxin.
  • a spectra may be generated from a sample that does not contain endotoxin to function as a baseline spectra. By comparing the baseline spectra with a spectra generated from a sample suspected to contain endotoxin, signals from impurities or other substances may be eliminated.
  • spectra obtained with known endotoxin standards as reference such as that from Escherichia coli 055.B5, Escherichia coli K-235, Klebsiella pneumonia, Pseudomonas aeruginosa 10, E. Coli 026 :B6, E. Coli 0127.B8, E. Coli 0128.B12, E. Coli 0111:B4, Salmonella typhosa, Salmonella enterica serotype Minnesota, Salmonella enterica serotype abortus equi, Salmonella enterica serotype enteritidis, Serratia marcescens, E. Coli J5(Rc mutant) and E.Coli F583(Rd mutant), so as to determine presence of the respective endotoxin. In some situations, absence of a signal may signify the absence of endotoxins.
  • detecting a signal from the enriched mixture may comprise identifying the endotoxin based on the signal and/or correlating an amount of the endotoxin with the signal.
  • a spectra of the sample suspected to contain endotoxin may be generated using laser induced fluorescence detector. By comparing the spectra obtained with that generated from known endotoxin standards as reference, endotoxins present in the sample may be identified. Amount of endotoxin present in the sample may also be calculated by correlating signal strength with that of the reference signals obtained with known amounts of the reference endotoxins.
  • kits for detecting and/or quantifying endotoxin may be developed based on the principles of the method as described above.
  • the kit may comprise a labeling agent capable of binding to endotoxin in a sample suspected of containing endotoxin to form a labeled endotoxin. Examples of suitable labeling agent have already been described above.
  • the kit may also comprise an enriching medium capable of enriching the labeled endotoxin in the sample.
  • the enriching medium comprises a sorbent selected from the group consisting of a solid phase extraction (SPE) packing material, silica- based aminopropyl sorbent, porous graphite column (PGC), carboxyl-coated magnetic beads, and combinations thereof.
  • SPE solid phase extraction
  • PLC porous graphite column
  • carboxyl-coated magnetic beads and combinations thereof.
  • the method and kit disclosed herein may be used in a myriad of industries such as biopharmaceutical industry, food processing, used water treatment, and water processing plants.
  • industries such as biopharmaceutical industry, food processing, used water treatment, and water processing plants.
  • water treatment and reuse of water are extremely important in the sustainable development of water resources.
  • Large numbers of microorganisms are likely to be present in used water and secondary effluents, and microorganisms are involved intensively in water treatment processes.
  • Bacteria may enter the pipe network, attach to the pipe wall and become part of a biofilm. Such bacteria can pose a potential threat to public health if endotoxins are produced. Removing these substances in reclaimed or reused water is therefore very important.
  • Monitoring of endotoxin concentrations during water treatment for example, can promote safety level of treated water.
  • endotoxins can also form aerosols and be suspended in the workplace atmosphere.
  • Workers at wastewater treatment plants may be exposed to an air mixture of microorganisms, microbiological components, chemicals, and gases, and may face the risk of a series of health effects, such as airway irritation and pulmonary diseases.
  • Worker exposure to endotoxins can manifest in both respiratory and general symptoms such as fever, coughing, irritation of the respiratory system, and chest congestion.
  • Associations between exposure of dust containing endotoxin and respiratory symptoms have been reported among sewage workers. It was suggested that long-term toxic pneumonitis reactions in chronically exposed workers may lead to irreversible decreased lung function and development of chronic obstructive pulmonary disease (COPD). Accordingly, monitoring of endotoxin levels is also important for maintaining work health safety at the work place.
  • COPD chronic obstructive pulmonary disease
  • Endotoxins are a class of macromolecules which may be released mainly by Gram- negative bacteria, and which may cause serious health problems.
  • Various embodiments disclosed herein relate to development of novel analytical methods for endotoxins, which are potentially toxic by-products of biopharmaceutical, food industry and sewage plants.
  • various embodiments relate to analysis of endotoxins in water by capillary electrophoresis - laser induced fluorescent detection (CE-LIF) and micro- extraction tip.
  • CE-LIF capillary electrophoresis - laser induced fluorescent detection
  • the analysis method may provide an efficient and reliable solution for the analysis of endotoxins concentrations using analytical instruments. Endotoxin extraction method was developed for reliable, robust analysis and interference removal.
  • embodiments disclosed herein may be used to monitor the endotoxin concentrations in both simple water matrices such as drinking water and medical solutions and complex water matrices such as reservoir water and waste water.
  • Labeling of the endotoxin may be conducted in the first step. Any label that can bind at any functional group of the endotoxin may be used.
  • fluorescent dyes fluorescein isothiocyanate (FITC) with an absorbance maximum at 495 nm and an emission maximum at 525 nm, were used to label endotoxins.
  • FITC fluorescein isothiocyanate
  • sensitive determination may be achieved by laser induced fluorescence detector, using 15 different endotoxin standards, including Escherichia coli 055.B5, Escherichia coli K-235, Klebsiella pneumonia, Pseudomonas aeruginosa 10, E. Coli 026.B6, E. Coli 0127.B8, E.
  • Coli 0128.B12 E. Coli 0111:B4, Salmonella typhosa, Salmonella enterica serotype Minnesota, Salmonella enterica serotype abortus equi, Salmonella enterica serotype enteritidis, Serratia marcescens, E. Coli J5(Rc mutant) and E.Coli F583(Rd mutant).
  • FITC-LPS was prepared by incubating LPS and FITC in borate or phosphate, bicarbonate, TEA, Bicine, CHES, CAPS, Glycine or Tricine solutions.
  • Solution pH value may range from 8.5 to 11.5, more preferably from 10 to 11.
  • Incubation was carried out for 1 h to 6 h at about 30 °C to 45 °C, more preferably 2 h to 4 h at 37 °C.
  • optimized ratio of FITC:LPS ranges from 1: 1 to 15: 1, preferably at around 3: 1.
  • the CE separation buffer may be one of borate or phosphate, bicarbonate, TEA, Bicine, CHES, CAPS, Glycine or Tricine buffers.
  • the pH value of CE running buffer may range from 8.0 to 11, preferably from 9.0 to 9.8. Concentrations of buffer may be 10 mM to 100 mM, more preferably at around 50 mM.
  • fluorescent dyes may also be applied for LPS labeling such as, but not limited to, Fluorescamine, FMOC, FQ, CBQCA, NanoOrange, Alexa 488 Hydrazide, NDA, Nile red, 5-DTAF, syto 9, syto 13, YO-PRO-1, TAMRA, RITC and Funl cell stain.
  • FIG. 13 shows the labeling of Alexa Fluor® 488 Hydrazide with LPS.
  • the calibration curve using LPS from E. Coli 055.B5 showed that the linear response ranged from 0.05 ppm to 100 ppm (FIG. 14). A detection limit of 50 ppb was obtained with the calculation based on signal-noise ratio (S/N) equal to 3.
  • purification or enrichment of the labeled molecules may be conducted by targeting any functional group that is present on the endotoxin.
  • the purification can be conducted via solid phase extraction.
  • the purification can be conducted by a carbohydrate or lipid extraction device.
  • sample pretreatment can be suitable for many different sample matrices.
  • the pretreatment method can extract target compounds of endotoxins from other interference materials.
  • it can be helpful to enrich endotoxins in the preparation procedures of final injection solutions and increase sensitivity of this analytical method.
  • FIG. 15 and FIG. 16 are the result comparison of LPS from Salmonella enterica serotype Minnesota using those techniques. Good recoveries were observed.
  • Solid phase extraction method allows the process with larger sample volume and therefore endotoxin enrichment effects.
  • Method sensitivity may increase 10 to 100 times to sub-ppb level.
  • Micro-extraction method may also be applied to remove excess fluorescent dye (FIG. 7). This method may be used to purify and prepare endotoxin standards after fluorescent labeling or other chemical modification.
  • endotoxin extraction method could also use lipid extraction device (for example, Lichrolut CN).
  • Various embodiments relate to (i) a method to detect and quantify endotoxins, comprising (a) labeling the endotoxin at a specific functional group on endotoxins; (b) purification/enrichment of endotoxins (LPS molecules) to remove interference from matrix effect; and (c) detection and quantification of endotoxins by an analytical instrument.
  • a method to detect and quantify endotoxins comprising (a) labeling the endotoxin at a specific functional group on endotoxins; (b) purification/enrichment of endotoxins (LPS molecules) to remove interference from matrix effect; and (c) detection and quantification of endotoxins by an analytical instrument.
  • kits to detect and quantify endotoxins comprising (a) a labeling reagent to bind at a specific functional group of the endotoxin; and (b) a purification/enrichment matrix or device to enrich endotoxins so as to remove interference from samples and excess labeling reagent after labeling.
  • LPS was derivatized with the amino-reactive fluorescent dye fluorescein isothiocyanate (FITC), separated by capillary zone electrophoresis (CZE) under the optimized conditions with the use of 50 mM sodium tetraborate buffer (pH 9.30), and detected by laser induced fluorescence LIF detector.
  • FITC fluorescent dye fluorescein isothiocyanate
  • CZE capillary zone electrophoresis
  • SPE solid phase extraction
  • Various embodiments disclosed herein provide high sensitivity and direct chromatography method for endotoxin assay. By way of the introduced extraction steps, interference from matrix effect and excess fluorescent dye is removed, and therefore is reliable. Extraction method can be applied to purification of LPS with fluorescent labeling or other chemical modifications.
  • embodiments disclosed herein may be present as an analytical kit giving direct analysis and peak visualization of endotoxin concentration based on peak area, and/or as a micro-extraction kit for interference removal and low concentration enrichment for endotoxin or other compounds with carbohydrate moiety.
  • Sodium dodecyl sulfates (SDS), sodium tetraborate, and sodium hydroxide were obtained from Merck (Darmstadt, Germany).
  • Fluorescein isothiocyanate (FITC), (1-3)- ⁇ - ⁇ - glucans were purchased from Sigma - Aldrich (St. Louis, MO, USA).
  • Lipopolysaccharides from Escherichia coli 0111:B4 (Lot # 024M4019V), Escherichia coli 055:B5(Lot #025M4040V), Escherichia coli 026:B6 (Lot # 053M4060V), Escherichia coli 0127:B8 (Lot # 103M4051V), Salmonella enterica serotype enteritidis (Lot # 064M4035V), Pseudomonas aeruginosa 10 (Lot # 100M4101V), Salmonella enterica serotype typhimurium (Lot # 093M4088V), Escherichia coli J5 (Rc mutant, rough strains) (Lot # 053M4112V), Salmonella typhosa Lot # 063M4017V), Escherichia coli F583 (Rd mutant, rough strains) (Lot # 055
  • Example 3 Labeling LPS with FITC
  • LPS was labeled with FITC by a protocol as described below. Briefly, FITC was dissolved in 50 mM sodium tetraborate buffer (pH 9.30). LPS solution was prepared by dissolving LPS powder in ultra-pure water. Then, FITC was added to LPS solution in the mass ratio of 2: 1 and incubated in dark at 37°C overnight, or at a higher temperature of about 30 to 60 °C for about 0.5 to 2 hours. The fluorescent dye was in excess amount to ensure complete labeling of LPS. FITC-labeled LPS was protected against light exposure throughout.
  • Solid phase extraction was performed using the 96-well MassPREPTM HILIC (hydrophilic interaction chromatography) ⁇ ⁇ plate (Waters) according to the manufacturer's protocols. Briefly, 100 ⁇ ⁇ LPS standards or samples were constituted with 90% (v/v) acetonitrile. Then the sorbent was conditioned with 200 ⁇ ⁇ of HPLC grade water and equilibrated with 2 x 200 ⁇ ⁇ of 90% (v/v) acetonitrile. LPS samples were loaded on the sorbent without vacuum, followed by sample washing twice with 200 ⁇ ⁇ of 90% acetonitrile. Finally, analytes were eluted with 100 ⁇ ⁇ of 1 mM Tris-citrate buffer.
  • Example 5 Capillary Electrophoresis
  • Fused-silica capillary were purchased from Polymicro Technologies Inc. (Phoenix, AZ, USA). Capillaries with an internal diameter of 50 or 100 ⁇ were used throughout. Prior to use, capillaries were rinsed with 1 M NaOH for 10 min, followed by rinsing with DI water for 10 min and separation buffer for 10 min. The samples were injected into the capillary by pressure injection for 5 s or 10 s under a pressure of 0.5 psi. The system was operated at a constant voltage of 30 kV. The sample storage plate temperature was set at 16 °C and the separation temperature was set at 25 °C. Separation buffer was refreshed after every five runs. Between runs, capillary was conditioned by rinsing with 0.1 M NaOH for 2 min, ultra- pure water for 3.0 min and separation buffer for 5 min.
  • Example 6 Water samples and sample pretreatment
  • FITC is an amine-reactive fluorophore, which reacts with primary and secondary amino groups to form thiourea. This reaction is sensitive to pH and temperature and the optimum reaction condition is reported to be at pH 9.0, and 20 °C to 25 °C. The derivatization reaction normally takes place for more than 12 hours due to the extremely slow kinetic.
  • Choice of the separation buffer plays an important role in the electrophoretic patterns of the analytes. Since the most commonly used buffers for the electrophoretic separation of carbohydrates are borate, this could also be applied for the separation of the LPS. Despite the fact that LPSs are neutral compounds, which makes them unsuitable for electrophoretic analysis, complexation of borate ions (B(OH) 4 " ) with cis-diol groups in the carbohydrate moiety within the LPS structure would enhance the electrophoretic mobilities of LPS. Borate complexation provides the uncharged glycolipid with negative charges thus making them mobile in CE analysis.
  • labeling reagent i.e. FITC
  • tris Tris(hydroxymethyl)aminomethane
  • DAB 1,4-diaminobutane
  • Example 12 Detection of different LPSs by capillary zone electrophoresis (CZE)
  • FIG. 4 shows the CE separation of 13 LPS standards under the optimized conditions.
  • the electropherograms of the LPS standards exhibit distinctive individual patterns. These characteristics are due to different charge-to- size ratios and heterogeneous structural properties of the individual components of LPS from a typical bacterial strain. However, there are no remarkable differences in the migration times. All the FITC-LPS peaks were detected at around 2 min. [00127] As there are many different bacteria strains in the environment and therefore numerous possibilities of LPS sources, it is impossible to confirm which bacteria generates certain particular LPS.
  • LPS from Escherichia Coli 055.B5 (a standard strain ATCC 12014) was used as the standard endotoxins in LAL assay, LPS from Escherichia coli 055.B5 was also used for quantification by the CE-LIF method as reference value. Endotoxin levels were calculated relative to the level of the reference endotoxin.
  • the stability of the derivatized LPS molecules in aqueous phase was also investigated.
  • the stability test was carried out during a period of 20 days using LPS from E. Coli 055 :B5, with the samples stored in dark at room temperature for 20 days. The samples were analysed each day to obtain consecutive electropherograms. The results showed that FITC itself was not very stable in aqueous solution at room temperature as a significant decrease in fluorescence signal was observed with longer storage times. In contrast, after labeling with LPS, the product exhibited high stability in aqueous solution at room temperature. No obvious degradation was found after 20 days (FIG. 5) of storage at room temperature. So the FITC labeling method should have great robustness in real analytical work.
  • Example 13 Solid phase extraction method to purify and enrich endotoxin
  • Selectivity of the stationary phase is an important parameter to be considered when the analytes are to be extracted from water.
  • solid phase extraction materials available commercially, covering a wide range of selectivity and applications.
  • the primary decision for trace analysis of endotoxin is the selection of the proper type of sorbent.
  • Silica-based aminopropyl sorbent has the capacity to strongly and selectively retain analytes that are hydrophilic in nature.
  • the retention of carbohydrates onto a HILIC stationary phase can be explained in terms of the hydrogen bonding as well as ionic and dipole-dipole interactions that occur while the carbohydrates partition into an immobilized water layer.
  • carbohydrate is one of the main components in the LPS structure, which is hydrophilic, the aminopropyl sorbent should be applicable to retain LPS.
  • solid-phase extraction (SPE) method specifically designed for capturing polysaccharides can remove most interference substances and cross-validate the analytical method.
  • SPE solid-phase extraction
  • peptide or protein can be labeled by FITC, but they will be removed during the SPE step; whereas polysaccharides can be captured by SPE, but they will not be labeled by FITC and therefore will not be detected by the CE-LIF method.
  • FIG. 6 indicates that LPS peaks were preserved after the SPE procedure, with recoveries were estimated to be approximately 70 %.
  • the SPE-CE-LIF method could avoid possible false positive results commonly associated with LAL assays, due to the reaction with (l-3)-P-D-glucans as these interference substances will trigger the LAL reaction.
  • the CE-LIF method provides inherent discrimination of interfering substances including (l-3)-P-D-glucans (FIG. 8), due to the specificity of the derivatizing agent.
  • Example 16 Quantitative data of SPE-CE-LIF method
  • Example 17 Determination of LPS concentration in water samples
  • a novel protocol for endotoxin analysis is disclosed herein by using HILIC ⁇ SPE plate to extract endotoxins from water samples, followed by pre-column FITC derivatization and a CZE separation coupled with LIF detector. It is well known that the identification and characterization of intact LPS, as amphiphilic macromolecular lipoglycan, is a complex and challenging area of research.

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Abstract

La présente invention concerne un procédé de détection et/ou de quantification d'endotoxine. Le procédé peut consister à mettre un échantillon susceptible de contenir de l'endotoxine en contact avec un agent de marquage pour former un mélange, la mise en contact étant réalisée dans des conditions suffisantes pour lier l'agent de marquage à l'endotoxine afin de former une endotoxine marquée, d'enrichir le mélange de l'endotoxine marquée et de détecter un signal provenant du mélange enrichi en tant qu'indication de la présence d'endotoxine. La présente invention concerne également une trousse de détection et/ou de quantification de l'endotoxine.
PCT/SG2017/050287 2016-06-06 2017-06-06 Procédé et trousse de détection et/ou de quantification d'endotoxine WO2017213588A1 (fr)

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